Biological motility, ranging from muscle contraction to intracellular vesicular to transport, can be attributed to the myosin molecular motor. At present, there are 15 distinct classes of myosin motors. At present, there are distinct classes of myosin motors. All myosins have the ability to convert energy from the hydrolysis of ATP into mechanical work as they cyclically interact with actin. Although much is known about myosin's mechanical performance, two major questions remain: 1) How is the chemistry of ATP hydrolysis coupled to force and motion generation at the molecular level 2) What is the structural basis for the mechanics of the myosin motor? To address these questions, we have developed a laser trap-total internal reflectance (TIRF) microscope? This instrument has the capacity to measure the force and generation generated by a single myosin molecule as it interacts with an actin filament, as well as simultaneously measuring fluorescence and polarization of a single fluorophore. Using the laser trap-TIRF microscope, we propose to determine: 1) How force and motion are coupled to specific steps in the hydrolysis of MgATP by measuring single myosin molecular motor displacements simultaneously with fluorescence data that indicate the presence of fluorescently-labeled MgATP and its hydrolysis products in the myosin active site; 2) What is the structural basis for the mechanics of the myosin motor? To address these questions, we have developed a laser trap-total internal reflectance (TIRF) microscope. This instrument has the capacity to measure the force and motion generated by a single myosin molecule as it interacts with an actin filament, as well as simultaneously measuring fluorescence and polarization of a single fluorophore. Using laser trap-TIRF microscope, we propose to determine: 1) How force and motion are coupled to specific steps in the hydrolysis of MgATP by measuring single myosin molecular motor displacements simultaneously with fluorescence data that indicate the presence of fluorescently-labeled MgATP and its hydrolysis products in the myosin active site; 2) If the myosin light chain binding domain acts a lever that rotates during the myosin powerstroke, then by fluorescently-labeling the regulatory light chain, we will be able to correlate fluorescence polarization measurements with unitary displacements as a means of determining whether or not the light chain binding domain rotates during the myosin powerstroke. In addition, the mechanics of Myosin V neck length mutants will be studied to test the lever arm hypothesis.